Single-Molecule Detection of Locus-Specific 8-Oxoguanine Using Hoogsteen-Guided Enzymatic Discrimination and Rolling Circle Transcription

Abstract 8-Oxo-7,8-dihydroguanine (OG) is a prevalent oxidative DNA lesion and a critical biomarker of oxidative stress, and its locus-specific quantification at single-base resolution remains challenging due to the low abundance of lesion. Herein, we report a locus-specific OG assay that integrates selective Hoogsteen base pairing, enzymatic discrimination, rolling circle transcription (RCT) and duplex-specific nuclease (DSN) with single-molecule counting. With target DNA present, it binds to a rationally designed linear template probe, leaving a single-nucleotide gap opposite the OG/G site. Subsequently, Bsu DNA polymerase selectively incorporates dATP opposite OG instead of opposite canonical G via Hoogsteen bonding, facilitating unambiguous base discrimination. The extended, fully paired linear template can be circularized by Taq DNA ligase through proximity ligation to generate a circular template for T7 RNA polymerase-driven RCT. The resulting long concatenated RNA transcripts can hybridize with the Cy5/BHQ2-labeled reporter probes, forming RNA-DNA duplexes that are specifically cleaved by DSN to liberate Cy5 fluorophore and intact RNA transcript. The RNA transcript can repeatedly initiate new cleavage cycles to achieve significant signal amplification. The liberated Cy5 fluorophores can be readily measured by single-molecule detection. This assay allows for highly selective and sensitive quantification of locus-specific OG, achieving a detection limit of 7.31 × 10−19 M and a broad dynamic range of 1 × 10−17to 1 × 10−11 M. It can reliably discriminate an OG frequency as low as 0.01% in excess unmodified DNAs, and accurately quantify the OG levels in genomic DNA under oxidative stress, holding great promise in oxidative DNA damage research and biomedical applications.